Abstract/Project Summary HIV-1 envelope glycoprotein (Env) is the sole target for development of antibody-mediated immune response. Studies of patients infected with HIV have led to the isolation of antibodies against different epitopes on the Env surface, capable of neutralizing a broad range of circulating HIV strains and providing passive protection against repeated viral challenges in non-human primates (NHPs). The current goal of HIV vaccine research is to create immunogens capable of eliciting similar antibody responses in nave individuals. Structure- based engineering of the soluble ectodomain of Env trimer is in the core of most of the current approaches. One major complicating factor with this approach comes from the large antigenic surface on the Env, where many off-target and competing epitopes are available to the immune system. The induction of immune responses against ?distracting? epitopes leads to attenuated and inconsistent response against the targeted bnAb epitopes of interest. NHPs are an important model for this work, because wildtype mice do not make Ab responses to major Env nAb epitopes observed in humans. Analysis of the polyclonal Ab response in NHP immunizations where soluble Env ectodomain trimers were used, revealed a set of highly immunogenic epitopes with low potential for the development of protective heterologous, and in some cases even autologous, responses. These epitopes include the base of the Env trimer ectodomain, N241/N289 glycan hole and variable loops (V1-V5). To address this shortcoming we have developed a rational approach to consistently elicit antibodies against the conserved fusion peptide (FP) epitope, for which there are several examples of human bnAbs. We have used our designer trimers and continuous antigen release program to generate preliminary results verifying success of this concept in NHPs. The reproducibility of the epitope targeting is the most impressive preliminary result presented here, and it indicates that reproducible elicitation of FP nAbs in humans may be readily achievable. Here we propose to conduct a series of experiments designed to further optimize this immunization strategy by minimizing the accessibility of immuno-distracting, off-target epitopes, and ultimately verify if protective, FP-targeting, bnAbs can be reproducibly elicited in NHPs. We will also incorporate our immunogens into multi-valent nanoparticles to improve immunogen trafficking, enhance B cell receptor avidity, and block the non-productive but highly immunogenic base epitope. Further, we will use continuous antigen delivery to improve the duration of germinal center reactions, and ultimately drive the high levels of somatic hypermutation necessary for generating bnAbs capable of neutralizing a wide diversity of HIV strains. This project will culminate by testing the efficacy of our vaccine regimen by challenging immunized NHPs with heterologous low dose repeated challenge.